R.A. Ledford

Identification of cell wall antigens associated with a large conjugative plasmid encoding phage resistance and lactose fermentation ability in lactic streptococci

In order to begin to analyze the gene products encoded by phage resistance plasmids in lactic streptococci, we identified phage-resistance plasmids by screening resistant strains from commercial starter cultures for the ability to carry out unselected cotransfer, by conjugation, of phage resistance with lactose fermentation ability (lac+). In this fashion, we identified a large (90 kilobases) plasmid, pCLP51R, that encodes the lac+ marker, resistance to a lytic phage called LP10G (1pr+), high-frequency conjugal donor ability (hft+), and clumpy growth of host bacteria in broth culture (clu+). The mechanism of resistance conferred by this plasmid appears to involve interference with a step in the phage replication cycle that occurs after the initial attachment of the phage. Polyacrylamide gel electrophoresis of cell surface extracts of isogenic strains, carrying or lacking pCLP51R, combined with immunoblotting analysis, showed that there were several plasmid-related differences in the banding pattern of low molecular weight proteins and that the plasmid resulted in production of several unique antigenic polypeptides in the size range of 15-30 kd, as well as modification of chromosomally encoded antigens to different molecular weight forms.

Microtiter plate assays for the measurement of phage adsorption and infection in Lactococcus and Enterococcus

Three easy and rapid microtiter plate assays for determining phage sensitivity of lactococci and enterococci have been developed. In the microlysis assay, the degree of sensitivity was measured on the basis of the ability of the bacterial cells to grow in the presence of various concentrations of phage and to effect a color change of an acid-base indicator as a result of acid production. Two assays that specifically measure phage adsorption to bacterial cells have been developed on the basis of the enzyme-linked immunosorbent assay (ELISA) technique. In the direct phage adsorption ELISA, adsorption of phage particles to cells immobilized onto microtiter plate wells was measured using specific anti-phage antibody. In the competitive phage adsorption ELISA, phage adsorption was assayed by allowing phage to compete with specific antibody binding to the bacterial cell surface. All three assays were quantifiable photometrically.

Protein characterization of lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages isolated from cheese wheys

Proteins of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages were studied using antibody inhibition assay and immunoblotting. Antisera were prepared against four representative L. lactis ssp. lactis and L. lactis ssp. cremoris phages (D59-1, F4-1, G72-1, and I37-1), which were selected from 17 isolates, derived from commercial cheese wheys. The reactivities of the four antisera with 13 other phage isolates were tested. Among these isolates, two phage groups having distinct serological properties were found. Group I reacted with the antisera against phages D59-1/F4-1 and Group II reacted with the antisera against phages G72-1/I37-1. Strongly lytic phages, capable of lysing phage-resistant host strains, were found to share protein similarities with the phage protein group I, and phages isolated from phage-sensitive host strains belonged to the phage protein group II. Furthermore, group I was composed of all prolate and some isometric phages, whereas group II was composed solely of the isometric phages. Thus, the two serologically distinct phage groups were not correlated with the two morphological groups, prolate and isometric. Proteins of the four phages were further characterized by immunoblotting and silver staining. A 22.5-kDa antigenic polypeptide of phage I37-1, and three polypeptides of 65, 37, 21 kDa in phage F4-1 were responsible for the cross-reactivities in group II and group I, respectively.